Previously, a novobiocin-hypersensitive mutant of Escherichia coli K-12 carrying a cysE-pyrE linked mutation, designated rfaD, which specifically affects the synthesis of the aldoheptose, L-glycero-D-mannoheptose, has been isolated and genetically characterized. The following facts have been established: (1) The truncated lipopolysaccharide (LPS) of the rfaD mutant contains D-glycero-D-mannoheptose rather than the normal L-glycero-D-mannoheptose. (2) The rfaD gene product, ADP-L-glycero-D-mannoheptose-6-epimerase, is required for the conversion of ADP-D-glycero-D-mannoheptose to ADP-L-glycero-D-mannoheptose. (3) The nucleotide ADP-D-glycero-D-mannoheptose accumulates in the rfaD strains. These results provide support for the following biosynthetic reactions: (a) the conversion of D-glycero-D-mannoheptose-1-P and ATP to ADP-D-glycero-D-mannoheptose and pyrophosphate and (b) ADP-D-glycero-D-mannoheptose is converted to ADP-L-glycero-D-mannoheptose. These two reactions are catalyzed by the enzymes ADP-D-glycero-D-mannoheptose-synthetase and ADP-L-glycero-D-mannoheptose-6-epimerase, respectively. The rfaD gene has been cloned into pBR322, and the rfaD gene product has been partially purified from plasmid amplified strains. Recently, we have isolated a new mutant carrying a cysE-pyrE linked mutation, designated rfa-2, which results in an increased permeability to hydrophobic agents and a heptose-less LPS structure. Further, we have demonstrated that the rfa-2 mutation is genetically distinct from the rfaD locus and the rfa-2 phenotype is not abolished by plasmid carrying the wild type rfaD allele. The rfaD gene product, ADP-L-glycero-D-mannoheptose-6-epimerase, is detectable in crude extracts of rfa-2 mutant strains. An automated sugar analyzer has been developed in our laboratory which allows us to follow directly the conversion of the sugar intermediates involved in the biosynthesis of L-glycero-D-mannoheptose.